Abstract
The present studies were undertaken to obtain further insight into the selectivities of the enzymes, i.e., phospholipase A2 and acetyltransferase, involved in platelet-activating factor (PAF) production upon stimulation of human polymorphonuclear leukocytes (PMN) and platelets. After appropriate stimulation of the cells in the presence of [3H]acetate the total PAF and analogs, i.e., 1-alkyl-2-acetyl-, 1-alkenyl-2-acetyl-, and 1-acyl-2-acetyl-glycero-3-phosphocholine were isolated by high performance liquid chromatography. The isolated mixture was subjected to treatment with phospholipase A1 to differentiate acetate incorporation into 1-ether linked and 1-ester linked species. The ratio of acetate incorporation into 1-ether linked vs 1-ester linked PAF analogs amounted to 13.8±1.0 and 1.3±0.1 for PMN and platelets, respectively. When compared to the ratio of 1-ether linked and 1-ester linked species in the diradylglycerophosphocholine precursors in each cell type, i.e., 1.13 for PMN and 0.22 for platelets, these data suggested a pronounced selectivity for the phospholipase A2 and/or acetyltransferase in the process of PAF production. When the experiments were repeated with cells that had been pretreated with phenylmethanesulfonylfluoride (PMSF) to block the acetylhydrolase, the most dramatic effects were observed on acetate incorporation into 1-acyl-2-acetyl-glycero-3-phosphocholine, which increased much more than that into 1-alk(en)yl-2-acetyl-glycero-3-phosphocholine. Under these conditions, the ratio of acetate incorporation into 1-ether linked vs 1-ester linked PAF analogs became 1.4±0.2 and 0.17±0.02 for PMN and platelets, respectively. These values are very close to the 1-ether linked vs 1-ester linked species in the diradylglycerophosphocholine precursors for PAF in the respective cell type. These data suggested that the selectivities of phospholipase A2 and/or acetyl transferase for etherlinked species, as observed in non-PMSF treated cells, are only apparent and caused by rapid degradation of the 1-acyl analog either before or after acetylation. In line with this interpretation, we demonstrated that 1-acyl-2-acetyl-GPC can be deacylated to water-soluble acetyl-GPC and GPC by sonicated PMN and platelets and that this deacylation is completely blocked in sonicates from PMSF-pretreated cells. In addition, evidence is presented which indicates that the enzyme responsible for deacylation may be a lysophospholipase.
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Abbreviations
- PMN:
-
polymorphonuclear leukocytes
- PAF:
-
platelet-activating factor
- 1-alk(en)yl-2-acetyl-GPC:
-
total 1-ether linked 2-acetyl-GPC, i.e., sum of 1-alkyl- and 1-alkenyl- species
- PMSF:
-
phenylmethanesulfonylfluoride
- acetyltransferase:
-
acetyl-CoA:lysoPAF acetyltransferase (EC 2.3.1.67)
- acetylhydrolase:
-
PAF-acetylhydrolase (EC 3.1.1.47)
- GP[14C]C:
-
glycero-3-phospho[14C]choline
- HPLC:
-
high performance liquid chromatography
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van den Bosch, H., Sturk, A., ten Cate, J.W. et al. Studies on the selectivity of enzymes involved in platelet-activating factor formation in stimulated cells. Lipids 26, 967–973 (1991). https://doi.org/10.1007/BF02536486
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DOI: https://doi.org/10.1007/BF02536486